The first organized French settlement in Acadia was founded in 1604 on an island in Passamaquoddy Bay, on the present U.S.-Canadian border, by Pierre du Gua de Monts and Samuel de Champlain. In 1605 the colony was moved to Port-Royal (now Annapolis Royal, Nova Scotia), and that settlement became the centre of Acadia’s future. Because the French claimed for Acadia lands that had also been claimed by England, the colony was continually contested by both nations. In 1613 Port-Royal was destroyed, and its inhabitants were dispersed by an English military expedition from Virginia.

In 1621 King James I of England (VI of Scotland) awarded the lands of Acadia to Sir William Alexander for the purpose of founding the colony of Nova Scotia. In 1632 his son King Charles I ceded Acadia back to France, and, under the Company of New France, a renewed period of French colonization followed. A bitter struggle for power broke out in 1636 between two of the leading French officials of the colony—a struggle that eventually resulted in a local civil war. Acadia was under English rule from 1654 to 1670 and then reverted again to French rule and remained basically under French control for the next 40 years.

On October 16, 1710, as part of the War of the Spanish Succession (1701–14), Port-Royal was captured by the British. The Treaty of Utrecht (1713) gave Nova Scotia to Great Britain but left Cape Breton Island and Île Saint-Jean (from 1799 Prince Edward Island) with France. In 1755 many French-speaking Acadians were deported by the British because of the imminence of war with France, the question of Acadian neutrality, and the possibility of revolt. Several thousand of them eventually settled in French-ruled Louisiana, where their descendants became known as Cajuns.

In 1763, at the conclusion of the French and Indian War (the North American phase of the worldwide war fought between France and Great Britain beginning in 1754), Île Saint-Jean and Cape Breton Island also formally came under British rule; the province of New Brunswick was separated from Nova Scotia in 1784.